Chemokines are secreted pro-inflammatory proteins of small dimensions (70-130 amino acids) mostly involved in the directional migration and activation of cells, especially the extravasation of leukocytes from the blood to tissue localizations needing the recruitment of these cells (Baggiolini M et al., 1997; Fernandez E J and Lolis E, 2002).
Depending on the number and the position of the conserved cysteines in the sequence, chemokines are classified into C, CC, CXC and CX3C chemokines. A series of cell membrane receptors, all heptahelical G-protein coupled receptors, are the binding partners that allow chemokines to exert their biological activity on the target cells, which present specific combinations of receptors depending from their state and/or type. The physiological effects of chemokines result from a complex and integrated system of concurrent interactions: the receptors often have overlapping ligand specificity, so that a single receptor can bind different chemokines, as well a single chemokine can bind different receptors.
Usually chemokines are produced at the site of an injury, inflammation, or other tissue alteration in a paracrine or autocrine fashion. However, cell-type specific migration and activation in inflammatory and immune processes is not the sole activity of chemokines, but other physiological activities, such as hematopoiesis or angiogenesis, appear to be regulated by certain of these proteins.
Even though there are potential drawbacks in using chemokines as therapeutic agents (tendency to aggregate and promiscuous binding, in particular), chemokines offer the possibility for therapeutic intervention in pathological conditions associated to such processes, in particular by inhibiting specific chemokines and their receptors at the scope to preventing the excessive recruitment and activation of cells, in particular leukocytes (Proudfoot A, 2000; Baggiolini M, 2001; Haskell C A et al., 2002).
Amongst chemokine receptors, CXCR3 (also known as G Protein-Coupled Receptor 9 or GPR9) is a membrane receptor which is highly expressed in IL-2 activated T cells (for example CD4+ CD8+ T lymphocytes), Natural Killer cells, B cells, and (at lower levels and/or in cell cycle-restricted manner) in other non-hemopoietic cell types, such as neurons, mammary gland cells, and proximal tubule cells.
The peculiarity of CXCR3 is that, unlike other chemokine receptors, it shows a reduced number of specific CXC chemokine ligands (CXCLs): CXCL9 (also known as Monokine Induced by Gamma Interferon, MIG, Small Inducible Cytokine Subfamily B Member 9, or SCYB9), CXCL10 (also known as Interferon-Gamma-Inducible Protein 10, IP-10, Small Inducible Cytokine Subfamily B Member 10, or SCYB10), and CXCL11 (also known as Interferon-inducible T cell Alpha Chemoattractant, I-TAC, Interferon-Gamma-Inducible Protein 9, IP-9, H174, beta-R1, Small Inducible Cytokine Subfamily B Member 11, or SCYB11).
These three chemokines not only have an affinity in the nanomolar range for CXCR3, but share other important features: many amino acids are conserved amongst their sequences, all lack the “ELR” motif at the amino-terminus, they are all induced by gamma-Interferon, and all seem to have a prominent role not only in leukocyte (Th1 cells) migration in relationship not only with inflammation and autoimmunity but also with graft rejection and ischemia. These activities have been demonstrated in animal models, such as knock-out mice and mice treated with antibodies specific for the chemokine or the receptor. For example, the administration of antibodies directed against the extracellular domains of CXCR3, or against its ligands, results in the specific inhibition of the inflammatory responses mediated by this receptor (WO 01/72334; WO 01/78708; WO 02/15932).
The prior art shows many evidences on the molecular mechanisms associated to the interaction between CXCR3 and its ligands, and on their importance for human physiology. When compared to CXCL9 and CXCI10, CXCL11 appears to be the most potent inducer of CXCR3-mediated activation, internalization and of transendothelial migration in human and mouse leukocytes (Cole K et al., 1998; Lu B, et al. 1999, Sauty A et al., 2001). The activity or the expression of these molecules can be considerably up-regulated and modulated in relationship to various pathological conditions, as shown in animal models or clinical samples associated to graft rejection (Meyer M et al., 2001), tubercolosis (Sauty A et al., 1999), transplant coronary artery disease (Kao J et al., 2003), HIV-1 replication (Lane B R et al., 2003), type 1 diabetes (Frigerio S et al., 2002), ulceration of intestinal epithelium (Sasaki S et al., 2002), microbial infection (Cole A et al., 2001), sarcoid granulomatous reactions (Agostini C et al., 1998), atherosclerotic lesions (Mach F et al., 1999), multiple sclerosis (Sorensen T et al., 1999; WO02/098346), cancer (Trentin L et al., 1999; Robledo M M et al., 2001), skin diseases (WO 02/43758; Flier J et al., 2001), nephropathies (Romagnani P et al., 1999), thyroid diseases (Romagnani P et al., 2002), brain or spinal cord injuries (WO 03/006045), and many other autoimmune or inflammatory diseases.
Moreover, it has also been observed that the proliferation of endothelial cells can be modulated by the interaction between CXCR3 and its ligands (Luster A et al., 1995; Romagnani P et al., 2001). CXCR3-binding CXC chemokines show an angiostatic activity on endothelial cells, which can be inhibited by anti-CXCR3 antibodies, suggesting a strict relationship between the activation of this receptor and the cell cycle regulation, at least in endothelium.
Studies on structure-activity relationships indicate that chemokines have two main sites of interaction with their receptors, the flexible amino-terminal region and the conformationally rigid loop that follows the second cysteine. Chemokines are thought to dock onto receptors by means of the loop region, and this contact is believed to facilitate the binding of the amino-terminal region that results in receptor activation. This importance of the amino-terminal region has been also demonstrated by testing natural and synthetic chemokines in which this domain is modified or shortened. This processing, following proteolytic digestion, mutagenesis, or chemical modification of amino acids, can either activate or render these molecules completely inactive, generating compounds with agonistic and/or antagonistic activity (U.S. Pat. No. 5,739,103; WO 02/59301).
These observations suggest that regulation of leukocyte recruitment during inflammatory or immune reactions is based on a combination of such agonistic and antagonistic effects, as shown for the CXCR3-binding CXC chemokines and many other chemokines (Loetscher P and Clark-Lewis I, 2001; Lambeir A et al., 2001). Thus, chemokines with specific modifications in the amino-terminal region are considered having therapeutic potential for inflammatory and autoimmune diseases (Schwarz and Wells, 1999).
As many other cell-signaling soluble molecules (interleukins, growth factors), chemokines show physiological interactions not only with cell receptors but also with glycosaminoglycans (GAGs), although with varying affinities. These negatively charged molecules are formed by disaccharide repeats (such as heparin, chondroitin sulfate, heparan sulfate, dermatan sulfate, and hyaluronic acid) and naturally occur on cell surfaces, in the extracellular matrix, or in the circulation. They can be present in isolated forms or linked to proteins (proteoglycans, or PGs) following the post-translational addition of GAGs at serine residues.
Chemokines, as the other GAG-binding proteins, have basic residues (mainly Arginine and Lysine) clustered in short portions of their sequence which are suitable for this purpose but such motifs are structured in different manner for each chemokine, or group of highly homologous chemokines. Some of these GAG-binding sites have been associated to specific consensus, such as BBXB motifs (where B represents a basic residue, and X any other residue) or other arrangements (Kuschert G et al., 1999; Proudfoot A et al., 2001).
The main consequence of this interaction is the aggregation of the chemokines, a state which is believed to provide a protection from proteolysis, as well as a mechanism for the controlled and gradient-generating release of the chemokines, participating to the recognition and to the presentation of chemokines to the receptors as oligomers (Hoogewerf A J et al., 1997; Kuschert G et al., 1999). The interaction with GAGs and the formation of these gradients has been clearly demonstrated for many chemokines, and the relative affinity has been measured. Therefore, it has been suggested that also the modulation of such interactions may represent a therapeutic approach in inflammatory disease (Ali S et al., 2001; Patel D et al., 2001).
Means to achieve a therapeutic effect on the basis of the GAGs-chemokines interactions known in the art involve the generation of GAGs analogs modulating the interaction between endogenous GAGs and chemokines (WO 94/20512), the use of heparanase for eliminating GAGs (WO 97/11684), the administration of chemokine-GAGs complexes (WO 99/62535), the modification of GAGs binding domain with polymers (WO 02/04015), or the substitution of residues involved in GAG-binding activity (WO 02/28419).
Even though extensive studies have been performed on some chemokines, it is well established that is not possible to anticipate, on the basis of the sequence homology with chemokine having limited similarity or known GAG-binding protein motifs, which specific basic residues have to be modified with non-conservative substitutions to impair GAG-binding, since there is a significant structural diversity of GAG-binding domains amongst the chemokine protein family (Lortat-Jacob H et al., 2002). Methods of detecting or identifying ligands, inhibitors or promoters of CXCR3 are also known in the art (U.S. Pat. No. 6,140,064). However, none of these approaches can be actually applied for generating and studying GAG-binding defective CXCL9, CXCL10, or CXCL11. Structural requirements for the interaction with GAGs, neither their tridimensional structure, are known for CXCR3 and for its ligands. There is no disclosure in the prior art of which may be the residues of these chemokines involved in GAG-binding, as well the in vivo effects deriving from their non-conservative substitution in mutant proteins.